Enzymes involved in the synthesis, utilization, and interconversion of phosphorylated metabolites require specific activation by inorganic cations. Reactions that involve metal-nucleotide complexes as substrates or effectors are of central importance in a wide range of cellular functions from metabolism to signal transduction. The goals of this project are to understand the relationship between the structures of enzyme-metal-substrate complexes and the basis for chemical activation of these molecules in enzymic catalysis. The research uses the spectroscopic methods of electron paramagnetic resonance, nuclear magnetic resonance, infrared, and synchrotron -x-ray spectroscopy, together with x-ray diffraction techniques, to determine structures of enzyme metal-substrate or inhibitor complexes with pyruvate kinase, creatine kinase, 3-phosphoglycerate kinase, and enolase. The specific aims of the project include: identification of the way by which pyruvate kinase exploits its three metal cofactors in activation of the transferrable phospho group of its substrates; the involvement of the metal cofactors in the activation of the keto add substrate for pyruvate kinase; high resolution x-ray crystallographic studies of crystalline complexes of rabbit muscle pyruvate kinase; studies of the roles of the two divalent cations at the active site of enolase in binding and activation of the substrate and in binding of inhibitors that mimic the carbanion intermediate in this reaction; an investigation of the thio acid analogue of 3-phosphoglycerate in activation of domain closure in 3phosphoglycerate kinase; and studies of linkage isomerism in ligands bound to the metal center in creatine kinase. Information on the structures of enzyme-substrate complexes is essential for understanding the fundamental chemistry of enzymic catalysis and for rational design of enzyme-specific inhibitors that can be used as therapeutic agents, or as biochemical and physiological reagents to manipulate, selectively, reactions or pathways.